The present invention relates generally to electroporation systems, and more particularly to providing electroporation systems with accurate, and even user settable, time constants.
It is known that exposure of cells or other biological molecules to intense electric fields for brief periods of time temporarily destabilizes membranes. This effect has been described as a dielectric breakdown due to an induced transmembrane potential, and has been termed “electroporation”. Among the procedures that use electroporation are the production of monoclonal antibodies, cell-cell fusion, cell-tissue fusion, insertion of membrane proteins, and genetic transformation.
The cells or tissue are exposed to electric fields by administering one or more direct current pulses. These pulses are administered in an electrical treatment that results in a temporary membrane destabilization with minimal cytotoxicity. The intensity of the electrical treatment is typically expressed in terms of the field strength of the applied electric field. This electric field strength is defined as the voltage applied to the electrodes divided by the distance between the electrodes. Electric field strengths used in electroporation typically range from 250 to 10,000 V/cm
For efficient electroporation, it is necessary to control the shape, e.g. time constant of the electrical pulse. For example, electroporation itself occurs within a narrow range of parameters, such as pulse voltage and pulse duration, which is exhibited by a narrow window between electrocution and little or no electroporation. If a pulse with too long a duration or too high a field strength is used, the cells may be lysed (destroyed). If the duration or field strength of a pulse is too low, electroporation efficiency is lost. As an added difficulty, the optimal voltage and time constant varies with the type of cell. The current emphasis on using electroporation to study cells that are sensitive and difficult to transfect (move molecules through membrane) makes the control of electroporation conditions particularly important.
One problem in selecting the electroporation parameters is that the sample itself (cells plus buffer) is a significant factor in the load imposed on an electroporation system, and therefore the time constant. Ragsdale I provides a description of how to measure a resistance of the sample. In theory, a parallel resistance can be set to allow delivery of a pulse with the proper time constant as described in Ragsdale II.
However, the resistance of the sample changes over time, which in turn affects the shape of the electrical pulse that is being applied. For example, the resistance of the sample before the electrical pulse is applied is different than the resistance after X seconds, which is different after further application of the electrical pulse. Thus, it is very difficult to achieve a desired shape of the electrical pulse.
It is therefore desirable to provide methods, systems, and apparatus for achieving a specified time constant, e.g. one that a user can dial-in (i.e. user settable).